Tumors are assemblies of cells that show excessive autonomous proliferation, and are classified into malignant tumors, which may result in death, and benign tumors; however, it can be difficult to discriminate between these types. Cancers are genetic diseases attributed to the mutation of genes such as oncogenes and tumor suppressor genes, and their causes can be found at a cellular level. Oncogene products constitute a signaling network within cells, and are involved in regulating signal transduction systems for cell division and differentiation. When their regulation becomes abnormal, cells do not differentiate further, but rather divide infinitely; that is, they become tumorigenic. If a way could be found to enable such an abnormal signal transduction pathway to be converted into a normal one, anticancer agents could be developed that would obtain excellent therapeutic effects without side effects.
At present, cancers are for the most part treated in three ways: with surgical therapy, chemical therapy, and radiation therapy. In practice, combinations of these therapies or further combination with laser therapy are prevalent. However, chemical therapy is preferred to other therapies when the pain accompanying therapy and metastatic conditions are taken into account. Numerous anticancer agents have been developed, most of which are based on the selective killing of cells that are actively dividing. However, these anticancer agents suffer from the disadvantage of also killing some normal cells, such as immune cells and hair root cells, with concomitant significant side effects; they are thus not able to be used for long periods of time. Therefore, there remains a need for novel anticancer agents. A therapeutic agent based on the causes of cancer might be expected to be highly effective and to have few or no accompanying side effects.
Abnormal activation of oncogenes induces cell proliferation, and is one cause of cancer. In contrast, tumor suppressor genes function to prevent abnormal cell proliferation or to trigger programmed cell death (apoptosis). Often, tumor suppressor genes trigger apoptosis to kill the cells with abnormally activated oncogenes thus preventing the formation of cancerous cells. Where tumor suppressor genes show normal activity, cells with abnormally activated oncogenes cannot progress toward cancer, but are annihilated. Therefore, to become cancerous cells, cells must have inactivated tumor suppressor genes as well as activated oncogenes.
One of the mechanisms by which tumor suppressor genes are inactivated is by hyper-methylation of CpG islands (Jones and Laird, Nature Genet. Vol. 21, 163-167, 1999). Methylation of CpG islands is performed by DNA methyltransferase. After significant methylation, DNA binding proteins such as methyl cytosine binding protein 2 (MECP2) bind to the methylated cytosine of the DNA, which recruits histone deacetylase (HDAC) to repress gene expression. In detail, HDAC removes the acetyl groups associated with histones, and the chromosomal DNA in the vicinity of the deacetylated histones becomes dense, which leads to repression of gene transcription. If gene expression is repressed by DNA methylation, DNA methyltransferase or HDAC inhibitors may be useful for inducing gene expression. Tumor suppressor genes whose expression is repressed by DNA methylation are exemplified by RB1, TP53, VHL, CDKN2A, CDKN2B, MLH1, and APC (Jones and Laird, Nature Genet. Vol. 21, 163-167, 1999).
As mentioned previously, histone acetylation and deacetylation are known to play important roles in regulating DNA transcription in eukaryotic cells (Grunstein M., Nature, 389, 349-352, 1997). Some naturally occurring compounds have been found to prevent cells from progressing toward cancer by inhibiting HDAC. Exemplified by trapoxin, trichostatin A, and depudecin, HDAC inhibitors have been studied for their ability to reverse the transformation of cancerous cells. Of the HDAC inhibitors, depudecin is the best characterized as to its anti-angiogenic activity in vivo and in vitro. In addition, the HDAC inhibitors have been studied with regard to cellular responses, including cell cycle interruption, alteration of gene expression patterns, and induction of apoptosis.
The TGF-β signal transduction system is well known for its tumor suppressor activity. The binding of TGF-β to TGF-β receptors causes the activation of the receptors, which in turn activate Smad proteins by phosphorylation. Once activated, Smad proteins move into the nucleus and regulate gene expression in cooperation with other transcription factors, thereby suppressing cell division or inducing apoptosis (Massague et al., Cell, 103 (2):295-309, 2000). Runx3 is one of the transcription factors that physically interact with Smad proteins (Hanai et al., J. Biol. Chem. 274; 31577-31582, 1999). Deletion or mutation of TGF-β receptors or Smad genes is observed in cells of various types of cancer. The tumor suppressor activity of TGF-β receptors was also demonstrated by an experiment in a cell strain lacking the TGF-β receptor. When the cell was transformed to express the TGF-β receptor, cell proliferation was reduced and tumorigenesis was decreased in an assay in nude mice (Chang et al., Cancer Res., 57 (14):2856-2859, 1997). The TGF-β signal transduction system is well characterized as to the repression of cell proliferation, which is achieved by promoting the expression of the CDK inhibitor protein p21. However, the mechanism by which TGF-β induces apoptosis remains to be clearly elucidated.
PEBP2 (polyoma virus enhancer binding protein 2) is composed of two submits, α and β. There are three genes which encode the α subunit.: RUNX1/PEBP2α B/CBFA2/AML1, RUNX2/PEBP2α A/CBFA2/AML2, and RUNX3/PEBP2α C/CBFA3/AML2 (Bae and Ito, Histol. Histopathol, 14(4):1213-1221, 1999). The RUNX1, RUNX2, and RUNX3 genes show homology of about 60-70% in amino acid sequence among them. They are highly conserved evolutionarily, with homology of about 95% between mouse and human.
Regarded as an important causative gene in leukemia, the RUNX1 gene becomes associated with other genes by chromosome translocation to cause acute myeloid leukemia or acute lymphoid leukemia in humans (Miyoshi et al., EMBO J., 12:2715-2721, 1993; Romana et al., Blood, 85:3662-3670, 1995; Okuda et al., Blood, 91:3134-3143, 1998; Okuda et al., Cell, 84:321-330, 1996). The RUNX2 gene plays a crucial role in osteogenesis, and its disruption has been implicated in causing cleidocranial dysplasia (Komori et al., Cell, 89:755-764, 1997; Lee et al., Nat. Genet., 15:307-310, 1997; Mundlos et al., Cell, 89:773-779, 1997; Otto et al., Cell, 89:756-771, 1997). Also, it has been reported that the RUNX2 gene shows oncogenic activity in the formation of T-cell lymphoma (Stewart et al., Proc. Natl. Acad. Sci. U.S.A., 94(16):8646-8651, 1997).
The RUNX3 gene was identified by the present inventors several years ago as a member of the PEBP2 family (Bae et al., Gene, 159(2):245-248, 1995; Levanon et al., Genomics, 23(2):425-532, 1994). However, diseases associated with the activation or inactivation of the RUNX3 gene, other than what we describe in this invention, hasn't been reported.